Ligands used for detecting prions

ABSTRACT

The invention relates to the use of specific ligand carriers for detecting prions. The invention further relates to substances that are capable of binding to prions and that are subsequently used to detect prions.

[0001] The present invention relates to the use of ligands for detectingprions, in particular in connection with a high speed BSE test.Moreover, substances are disclosed which are suitable for binding andthe subsequent determination of prions.

[0002] Prions are normal cellular proteins (PrPc) which can be detectedabove all on the surface of neurons. By conversion of the normalcellular protein into its abnormal isoform (PrPsc), a pathogenic formwith new properties is formed. In contrast to the normal form, the formthus obtained is not water-soluble and cannot be degraded by proteases.According to the so-called “protein only” hypothesis, the simultaneousoccurrence of pathological and normal prions results in the normalmolecule changing its conformation and adopting the pathological form.This process leads to the accumulation of these proteins in the brainand finally to encephalopathies which include the Traber disease(scrapie) in sheep, the bovine spongiform encephalopathy (BSE) in cattleand the Creutzfeldt-Jakob disease (CJD) in man.

[0003] Glycosaminoglycans (GAGS) are linear heteropolysaccharides whichare anchored to the cell surfaces and play a major role in cell adhesionand migration. The intracellular insertion of prions via these moleculesis also possible.

[0004] Pentosan (poly-b-xylose-2,3-disulphonate; pentosanpolysulphonate) is a polysulphonated polyglycoside (molecular weight4,000 to 12,000 Dalton). This molecule seems to inhibit the accumulationof prions. It has been shown that, in comparison with other sulphonatedGAGS such has heparin, heparan sulphate and chondroitin sulphate, thissubstance inhibits the production of prions most strongly. Thisinhibition may be attributable to a direct linkage with the prions [B.Caughey and G. Raymond, Journal of Virology, February 1993, p. 643-650;B. Caughey et al, Journal of Virology, April 1994, p. 2135-2141; D.Sawitzky, Med. Microbiol Immunol. (1996) 184: 155-161; R. Gabizon et al,Journal of Cellular Physiology 157: 319-325 (1993); Show-Ling Shyng etal., Journal of Biological Chemistry, 1995 by the American Society forBiochemistry and Molecular Biology, Inc., C. Farquhar et al, The Lancet,Vol. 353, Jan. 9, 1999].

[0005] PrP-specific monoclonal and polyclonal antibodies have alreadybeen produced which recognise both variants PrPc and PrPsc. By means ofthe monoclonal antibodies available, the two variants can bedifferentiated indirectly and a disease detected in this way.

[0006] This has so far been done by mixing the samples to be examinedwith proteinase K which destroys only PrPc but not PrPsc. By means ofelectrophoresis, both proteins are separated and the pathological formis detected with antibodies. Although the sensitivity and specificity ofthis test is high, not all cases of BSE can be detected since a positiveresult in these tests depends on the total quantity of the prions usedfor the investigation. However, a sufficiently high overall amount ispresent in the brain tissue as a rule only during the final stage of thedisease so that, so far, it has been possible to carry out thecorresponding investigations in a standard manner only on tissue fromdead beings (R. Meyer, Dt. Ärzteblatt 97, Vol. 49, Aug, 12, 2000, pageA-3314). Moreover, these tests based on electrophoresis aretime-consuming and expensive.

[0007] Recently, it has been possible to track down the pathogens alsoin the blood of sheep and moose by means of organic solvents and aspecial type of chromatography and to detect them by means of a Westernblot test [M. Schmerr et al., Journal of Chromatography A, 853 (1999)207214]. However, such a test is extremely time-consuming and expensiveand hardly feasible in practice as routine test.

[0008] Adriano Aguzzi et al. recently described PrPsc as bindingspecifically to plasminogen [M. B. Fischer et al, NATURE, Vol. 408, 23.November 2000]. A test based on this binding was not presented.

[0009] It has thus not been possible so far to find specific laboratorymarkers for these diseases and, as a rule, the diagnosis is possibleonly after the death of the patient or after the animal has been killed[S. B. Prusiner, PNAS USA, Vol. 95, pp. 13363-13383, November 1998; A.L. Horwich and S. Weissman, Cell, Vol. 89, 499-510, May 1997].

[0010] In DE 197 30 132 A1 and WO 00/29850, in vitro methods for thedetection of BSE pathogens in a specimen are described; however, incontrast to the method according to the invention, the detection iscarried out directly by means of immobilised antibodies. The use ofglycosaminoglycan is not described.

[0011] Until the present invention was made, it had not been possible toimplement any process commercially by means of which any BSE diseasemight have been detectable in living organisms. However, it would beprecisely this which—in view of the present incurability of this diseaseand the proven transferability of the disease from infected animals(e.g. cattle) in which the disease need not yet have broken out,however, to man via the food consumed—would be of paramount importancefor the comprehensive prophylaxis and for the successful elimination ofthe disease. In this connection, the economic losses caused by thisdisease amounting to billions also deserve to be mentioned.

[0012] In spite of the extremely high need for a BSE test overcoming theabove-mentioned disadvantages, which necessarily arises from the detailsstated above, it has not been possible, up until the present invention,to provide such a test.

[0013] In view of the above-mentioned state of the art, the presentinvention is consequently based on the task of providing a method bymeans of which BSE pathogens (PrPsc) can be specifically detected. Thismethod should be easy and rapid to handle and safe and cost-effective toexecute and permit an extremely sensitive detection. Moreover, it shouldbe possible to carry out this method in vitro on samples with low PrPscconcentrations which can be obtained from living organisms, for example.

[0014] This task and others not explicitly mentioned which neverthelesscan be directly derived or deduced from the contexts discussed above areachieved by way of a method which has all the characteristics of claim1. Appropriate variations of the process according to the invention areprotected in the sub-claims which refer back to claim 1.

[0015] By treating, in an in vitro process,

[0016] (i) a sample with proteinase K

[0017] (ii) bringing the sample thus treated into contact with a bindingprinciple which is bound to a solid carrier

[0018] (iii) separating the binding principle bound to the solid carrierfrom the sample and

[0019] (iv) detecting the BSE pathogen (PrPsc) which, if necessary, isbound to the binding principle and originates from the sample, whereby

[0020] (v) the binding principle (ii) is selected from the groupconsisting of: a glycosaminoglycan, fibronectin or lipoprotein A,

[0021] it is possible to make a process available in a not directlyforeseeable manner for detecting BSE pathogens in a sample, whichprocess allows this detection to take place in a simple manner. In thisconnection, this process exhibits the above-mentioned advantagescompared with the state of the art.

[0022] In particular, it is

[0023] simple and rapid to handle and

[0024] safe and cost-effective to execute and it can

[0025] be carried out on samples originating from living organisms suchas blood samples, tissue samples or body fluids such as urine, milk,liquor or saliva or

[0026] on soil or plant specimens

[0027] According to the invention, BSE pathogens are detected. Thisshould be understood to mean the pathological form of the prion protein(PrPsc).

[0028] For the degradation of the non-pathological prion protein (PrPc),proteinase K is added to the sample. It is known from the state of theart that proteinase K hydrolytically splits the non-pathological formbut the pathological form of the prion protein only to a small extent.In view of the present invention, however, it is clear to the personsskilled in the art that other proteases which satisfy theabove-mentioned purpose can also be used according to the inventionwithout deviating from the scope of the present invention. Thehydrolytic splitting processes using proteinase K are well known to thepersons skilled in the art. In particular, a series of operatingprocedures are contained in laboratory handbooks in which the buffersystems suitable for use, the incubation periods to be applied and otherreaction conditions to be satisfied are described in detail.

[0029] The hydrolytic degradation of the non-pathological prion proteinwith proteinase K is described by M. Schmerr et al, in Journal ofChromatography A, 853 (1999) 207-214, for example.

[0030] It is, for example, possible to degrade the PrPc by subjecting asample to a 30 minute degradation by proteinase K in a 250 μg/mlproteinase K solution in a commonly used Tris/EDTA buffer, pH 7.4, 37°C.

[0031] The prion proteins which are not hydrolytically split and which,according to the invention, represent the pathological PrPsc form areremoved from the sample by means of a binding principle. This bindingprinciple consists preferably of a glycosaminoglycan, particularlypreferably pentosan polyphosphate. Other preferred glycosaminoglycanswhich can be used according to the invention are: heparan sulphate,heparin, hyaluronic acid, chondroitin sulphate, dermatan sulphate,keraton sulphate or Congo Red. Similarly preferably usable according tothe invention is an artificially produced sulphonated glycosaminoglycan.Fibronectin can also be preferably used as binding principle.Lipoprotein A can also be preferably used as binding principle.

[0032] According to the invention, this binding principle is bound to asolid carrier.

[0033] Preferably, all solid carriers which are known to the personsskilled in the art in the field of affinity chromatography can beconsidered for use as solid carrier. Particularly preferably, so-calledbeads are used according to the invention, i.e. usually sphericalmicro-carriers of different materials.

[0034] Other solid carriers which can preferably be used according tothe invention are those column materials which have found widespread usein affinity chromatography.

[0035] Microtiter plates coated with the binding principle describedabove are also preferably used according to the invention.

[0036] Nanobeads containing europium (Eu), from Seradyn, Indianapolis,USA, are particularly preferred. These particles are “absorbed” witheuropium chelates in such a way that the surface remains free forcoupling reactions and the europium chelates do not escape. Europiumchelate: tris(naphthyl trifluorobutanedione) Eu. When these particlesare stimulated with UV light (333 nm maximum), they emit light radiationat 613 nm with a duration of approximately 0.5 milliseconds which is10,000 to 100,000 times longer than the emission period of mostflurophores. This extremely long emission period and the large Stokesdisplacement (difference between emission and excitation wavelength)permits their use in the tests based on time-delayed fluorescence. Eachparticle contains >30,000 europium atoms in tris-naphthyltrifluorobutanedione (a diketone). For the 100 nm particles, theso-called quantum yield is equivalent to approximately 3,000 moleculesof fluorescein (one of the most widely used fluorophores).Phycobiliprotein (probably the most strongly fluorescent compoundknown), in comparison, has a quantum yield corresponding to that ofapproximately 30 fluorescein molecules. Since a 100 nm particle has adiameter approximately 10 times bigger than the phycobiliprotein and a1000 times larger volume mass ratio, these beads have a fluorescencewhich, on a molar basis, is 100 times higher than that ofphycobiliprotein.

[0037] The nanoparticles are first irradiated (the so-called excitationwavelength for europium is 340 nm) and, after a delay of 400 μs (in thiscase), the signals are measured for a length of 400 μs. As a result ofthis delay, registering non-specific signals of short duration from thematrix is avoided. The results are recorded in RFU (relativefluorescence units). The measuring operation takes place automaticallyby means of a standardised fluorimeter which can be used to measure timedelayed fluorescence.

[0038] By means of the test system according to the invention,investigations are successfully carried out on samples originating fromliving organisms, such as blood samples, tissue samples or body fluidssuch as urine, milk, liquor or saliva for the presence of BSE pathogens(PrPsc; pathological form of the prion protein).

[0039] It is also possible to carry out investigations into soil orplant specimens.

[0040] Particularly preferably, thrombic starting material subjected tolysis is used as sample. For this purpose, a blood sample is caused toclot and the thrombic material thus formed is separated off. Thethrombic material is degraded by hydrolysis with a proteinase and theresulting sample is used directly in the test.

[0041] The BSE pathogen bound to the binding principle is preferablydetected by specific antibody tests. These tests and the method ofdetection are also well known to the persons skilled in the art. ELISAand RIA methods as well as agglutination tests and flow cytometry can bementioned as examples.

[0042] In principle, all processes can be applied which permit detectionvia a specific initial antibody, if necessary by using a second antibodyspecific for the first antibody. Methods which include a reinforcingsystem such as an enzyme coupled to a first or second antibody can beused particularly preferably according to the invention.

[0043] Specific antibodies have already been described and can bepurchased from Prionics AG, University of Zurich, Switzerland.

[0044] A system particularly preferred according to the invention forthe detection of the PrPsc bound to the binding principle comprisesantibodies covalently bound to nanoparticles with a diameter of 10-100nm. The nanoparticles contain europium. By means of time-delayedfluorescence, these particles and consequently indirectly the antibodiesor the binding of the antibodies to PrPsc or to the first antibody canbe detected highly sensitively.

[0045] Moreover, it is preferred for the antibodies of theabove-mentioned paragraph to be bound to the nanobead not covalently butvia the biotin/streptavidin system which is well known to the personskilled in the art, the antibody being preferably present in thebiotinylated state.

[0046] A further preferred practical example of the present inventioninvolves the carrier being present in the labelled state for thedetection, in particular in the form of a Eu labelling (Eu nanobeads)and, following depletion from the sample by binding to a second bindingprinciple, in particular a specific anti-PrPsc antibody, detecting thecorresponding labelling by the corresponding detection, in particulartime-delayed fluorescence.

[0047] A characteristic feature of the present invention is that theindividual aspects of the detection process according to the inventionare known to the persons skilled in the art. However, it was impossibleto foresee in any way that a combination of these individual steps wouldlead to a method which, in such a highly surprising and favourablemanner, provides a simple solution to such intensive needs of thepersons skilled in the art.

[0048] The following examples provide a more detailed explanation of theinvention. However, they should not be understood to be limiting in anyway.

EXAMPLE 1

[0049] Binding of Pentosan to Microcarriers.

[0050] Toyopearl HW55 (commercially available cross-linked polyacrylategel from Toyo Soda Manufacturing Co. Ltd. with a particle size of 50-100μm) was used as the carrier.

[0051] 6 ml of a saturated aqueous NaOH solution and 15 mlepichlorohydrin were introduced into 10 ml of the gel and the reactionmixture was incubated for two hours with stirring at 50° C. The gel waswashed in sequence with alcohol and water in order to introduce epoxygroups into the gel. 20 ml concentrated aqueous ammonia were added tothe resulting epoxy-activated gel and the reaction mixture was stirredfor two hours at 50° C. in order to introduce amino groups into the gel.

[0052] 3 ml of the activated gel thus obtained which contained aminogroups were added to 10 ml of an aqueous solution containing 200 mgpentosan polysulphate (pH 4.5). 200 mg1-ethyl-3-(dimethlaminopropyl)-carbodiimide were added to the resultingreaction mixture, the pH of the reaction mixture being adjusted to 4.5and the resulting reaction mixture was shaken for 24 hours at 4° C. Oncompletion of the reaction, the resulting reaction mixture was washed insequence with 2 M aqueous NaCl solution, 0.5 M aqueous NaCl solution andwater giving the desired gel on which pentosan polyphosphate wasimmobilised.

EXAMPLE 2

[0053] Prion-containing test samples were treated according to the usualmethods with proteinase K to eliminate the normal prion proteins PrPc.

[0054] For this purpose, the sample was adjusted to a concentration of50 μg/ml proteinase K (Boehringer) and incubated for 1 hour at 37° C.Further details regarding the conditions for the enzymatic degradationare described by Schmerr et al, as above.

[0055] For example, the following protocol can be used:

[0056] 50 μl 1 mg/ml proteinase K (Sigma, catalogue no. 82456) aretransferred with a pipette into 1 ml of sample.

[0057] Incubation at 37° C. is carried out for 30 minutes.

[0058] 100 μl 4 mM Pefabloc SC PLUS (Roche; catalogue no. 1 873 601) areadded. This is followed by incubation for 5 minutes at room temperature.

[0059] To the supernatant liquor, beads coated with pentosanpolyphosphate from example 1 are added. It has been found that thepresence of 1 mM zinc promotes binding.

[0060] The beads are sedimented by centrifuging in a Hettich tablecentrifuge and separated from the test sample.

[0061] Subsequently, the beads are incubated with specific antibodiesagainst prions (Prionics AG, University of Zurich, 8057 Zurich,Switzerland) and tested in ELISA by means of a secondary antibody. Apositive test batch is characterised by increased addition of thesecondary antibody to the ligand complex which in turn can be measuredby the conversion of a colourless substrate by the enzyme bound to thesecond antibody.

[0062] It was possible to detect the BSE pathogen in the blood ofinfected animals in this way in a surprisingly simple manner.

EXAMPLE 3

[0063] Instead of an Elisa test, an agglutination test was carried out.

[0064] In this way, too, it was possible to detect the BSE pathogen in asurprisingly simple manner.

EXAMPLE 4

[0065] Instead of an Elisa test, the beads labelled with the secondantibody were detected by means of flow cytometry.

[0066] In this way, too, it was possible to detect the BSE pathogen in asurprisingly simple manner.

[0067] This procedure for detecting prions is entirely new andpracticable. By means of this method, blood samples, tissue samples,body fluids (e.g. urine, milk, liquor, saliva etc.) of animal, human andplant origin can be tested. Soil specimens, too, can be tested forcontamination.

EXAMPLE 5

[0068] For covalently binding antibodies to nanobeads (FLUOROMAX™fluorescent microparticles, Seradyn, Indianapolis, USA) these aretreated with carbodiimide and hydroxysuccinimide in order to ensure asecure and firm binding between the carboxyl groups on the particlesurface and the protein molecules (antibodies). In this way, theantibodies remain stably bound to the nanoparticles (longer useful lifeof the conjugate). The antibodies cannot be separated from the beadsduring the subsequent treatments (different reaction buffers).

EXAMPLE 6

[0069] Detection of the Binding of PrPsc to Pentosan Polyphosphate byMeans of Antibody-Europium-Nanobead Complexes.

[0070] The europium nanoparticles were purchased from Seradyn,Indianapolis, USA. These particles are “absorbed” with europium chelatesin such a way that the surface remains free for coupling reactions andthe europium chelates do not escape. Europium chelate:tris(naphthyltrifluorobutanedione) Eu. When these particles arestimulated with UV light (333 nm maximum), they emit light radiation at613 nm with a duration of approximately 0.5 milliseconds which is 10,000to 100,000 times longer than the emission period of most flurophores.This extremely long emission period and the large Stokes displacement(difference between emission and excitation wavelength) permit their usein the tests based on time-delayed fluorescence. Each particlecontains >30,000 europium atoms in tris-naphthyl trifluorobutanedione (adiketone). For the 100 nm particles, the so-called quantum yield isequivalent to approximately 3,000 molecules of fluorescein (one of themost widely used fluorophores). Phycobiliprotein (probably the moststrongly fluorescent known compound), in comparison, has a quantum yieldcorresponding to that of approximately 30 fluorescein molecules. Since a100 nm particle has a diameter approximately 10 times bigger than thephycobiliprotein and a 1000 times larger volume mass ratio, these beadshave a fluorescence which, on a molar basis, is 100 times higher thanthat of phycobiliprotein.

[0071] The nanoparticles are first irradiated (the so-called excitationwavelength for europium is 340 nm) and, after a delay of 400 μs (in thiscase), the signals are measured for a length of 400 μs. As a result ofthis delay, registering non-specific signals of short duration from thematrix is avoided. The results are recorded in RFU (relativefluorescence units). The measuring operation takes place automaticallyby means of a standardised fluorimeter which can be used to measuredtime delayed fluorescence.

[0072] This system is described by Ci, Y., et al., J. Immun. Meth, 179,233-241, 1995 as well as Souka et al. Clin. Chem., 47:3, 561-568 amongothers.

[0073] For similar systems (Eu-nanobead conjugates) detection limits forprostate-specific antigens of 7.3×10⁵ molecules/ml are reported (forprion protein, this would correspond to approximately 36.3 fg). The timedetection methods previously described are capable of detecting 50 pgprion protein (Barnard, G. et al., Luminescence, 15:6, 357-362, 2000).

EXAMPLE 7

[0074] Antibody Coupling to Carriers with the Biotin/StreptavidinSystem.

[0075] Biotin is used in the so-called two step techniques in connectionwith conjugated or immobilised strept(avidin). The binding of the biotinto the strept(avidin) takes place very rapidly and in a stable manner.Different techniques for the biotinylation of antibodies have beendescribed. Moreover, kits are available from different companies withthe corresponding protocol.

[0076] Normally, the biotin is conjugated by the primary amines of theproteins (e.g. lysine) and, in this way, 3 to 6 molecules of biotin arebound per protein molecule. As an example, sulpho-NHS-LC-biotin (Pierce)can be used. The separation of the non-conjugated biotin from theantibody can take place by means of NANOSEP™ centrifugemicro-concentrators (Pall). The biotinylation stage is determinedphotometrically by the so-called HABA reaction(HABA-2-(4′hydroxyazobenzene)-benzoic acid) with excess avidin.

[0077] Coating of the nanoparticles with streptavidin is carried outaccording to Härmä et al, Clin. Chem., 47:3, 561-568 (2001). Byprewashing the beads with PBS, pH 7.0 with NANOSEP™ centrifugemicroconcentrators, they are resuspended in the same buffer withultrasonic probe. Subsequently, the activation of the carboxyl groupswith 10 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDAC),(Pierce) and N-hydroxysulphosuccinimide (NHS), (Sigma) is carried outfor 30 minutes. After two wash processes (as above but with carbonatebuffer, pH 9.0), 15 μM streptavidin is added and the beads are incubatedfor 1 hour. At the end, the nanoparticles are washed 5 times with 2 mMtris-HCl (Sigma), pH 7.0, and stored at 4° C.

EXAMPLE 8

[0078] Binding of PrPsc to Europium Nanobeads Via Fibronectin (CompareExample 7).

[0079] It has been found in this case that it is particularly importantto use a method in the case of which the conformation of the fibronectinmolecules is influenced as little as possible.

[0080] The separation of the (nano) beads from the sample can take placeby centrifuging and/or by using so-called centrifuge concentrators(microfilters). Depending on the strength of the bond betweenfibronectin and PrPsc, the beads-PrPsc complexes can be washed in orderto achieve a maximum possible separation of the two PrP forms.

[0081] In principle, the procedure was as follows:

[0082] 1. Fibronectin is coupled with nanobeads (europium nanobeads)

[0083] 2. The sample is incubated with fibronectin nanobeads. Thepathological form of the PrP (PrPsc) adheres to the fibronectin and thenormal form (PrPC) does not—remains dissolved in the reaction mixture.

[0084] 3. Subsequently, the fibronectin nanobeads are separated from thesample by centrifuging and resuspended in a reaction buffer.

[0085] 4. The resuspended fibronectin nanobeads are incubated on amicrotiter plate coated with monoclonal antibodies (against PrP)(formation of the MoAk-PrPsc-fibronectin-nanobeads complex).

[0086] 5. After the wash process, the fluorescence is measured (timeresolution) and the concentration of PrPsc is determined.

[0087] Detailed instructions are as follows:

[0088] Human FN (FIBRONECTIN batch: 000832, BD Biosciences. MW: 440 kDa)or other fibronectin recombinants.

[0089] 1 mg/ml FN: one bottle of 1 mg FN is brought to room temperatureand dissolved in 1 ml sterile distilled H₂O for 30 minutes.

[0090] 0.5 ml 1% MP (Seradyn, FLUORO-MAX fluorescent particles Part no.1347-0350) is washed 2× with 0.5 ml 50 mM pH 6.1 MES (morpholinoethanesulphonic acid, Sigma product no. M8250) and NANOSEP 300 kDa cut-off(Pall Life Sciences; product no. OD300C33) for 5 minutes at 14,000 G.

[0091] After the last wash process, resuspension is carried out in 0.5ml 50 mM pH 6.1 MES, the suspension is transferred into 2.0 ml Eppendorftubes and these are filled up to 1.0 ml with 50 mM MES, pH 6.1

[0092] Preliminary Activation

[0093] a) EDAC (Carbodiimide—Pierce, Cat. No. 22980): 0.6 molar excess,0.5 ml 1% MP=5 MP; MP acid content meq/g=μmole/mg;

[0094] b) (acid content, μmole/mg) (5 mg MP) (desired ratio)=μmole EDACrequired: 0.1566×5×0.6=0.4698 μmole EDAC required

[0095] c) (μmole EDAC required)/(52 μmole/ml)=ml EDAC stock solution perml reaction: 0.4698/52=0.009 ml EDAC stock solution

[0096] d) EDAC stock solution: 10 mg/ml in distilled water, to beprepared directly before use.

[0097] e) Add with a pipette to the washed MP in the following sequence:

[0098] 230 μl NHS stock solution (50 mg/ml in distilled waterN-hydroxysuccinimide, Sigma, cat. No. H7377)

[0099] 9 μl EDAC stock solution

[0100] Incubate for 30 minutes at room temperature with continuousmixing.

[0101] Wash MP twice with 50 mM pH 6.1 MES and NANOSEP as in 3.

[0102] After the last wash process LWV, resuspend in 0.5 ml 100 mM pH6.1 MES and transfer into a 2.0 ml Eppendorf tube.

[0103] Add 0.5 ml FN solution from 2, mix thoroughly

[0104] Incubate for 2 hours at room temperature with continuous mixing

[0105] Wash MP twice with 50 mM pH 6.1 MES and NANOSEP as in 3.

[0106] After LWV, resuspend in 0.5 ml MNTA (25 mM MOPSO, Sigma cat. No.M8389, pH 7.4; 100 ml mM NaCl, Sigma cat. No. 71378; 0.1% TWEEN 20,Sigma cat. No. 27, 434-8; 0.1% NaN3, Sigma cat. No. 71290)

[0107] Store at 4° C.

[0108] Coating of the Microtiter Plate

[0109] Coat the plate (Nunc, Fluoro-Nunc modules F16 Maxi-Sorp, cat. No.47515)

[0110] 6H4 (monoclonal antibody, Prionics): 8 μg/ml, 96 wells—dilute inbic/carb buffer pH 9.4 (Pierce, cat. No. 28382), transfer 50 μl/wellwith a pipette

[0111] Incubate for 3 hours at 37° C.

[0112] Blocking of Unspecified Binding Sites

[0113] Wash 1×200 μl with wash buffer (5 mM TRIZMA pH 7.8, Sigma cat.No. 93349; 150 mM NaCl; 0.05% Tween20)

[0114] Introduce 100 μl blocking buffer (50 mM TRIZMA pH 7.8, Sigma cat.No. 93349; 150 mM NaCl; 2% bovine serum albumine, Immucore cat. No.004410; 2 gelatine Sigma cat no. G7765; 0.05% TWEEN 20; 0.05% NaN3) witha pipette per cavity.

[0115] Shake for 1 hour at room temperature—450 rpm

[0116] Suck off supernatant liquor.

[0117] Incubation of Antigen MP-FN

[0118] Dilute MP-FN 1:742 in PBS-TWEEN 20 2% (PBS Pierce cat. No. 28374;Tween20 2%)

[0119] Incubate specimen treated with 50 μl proteinase K in Eppendorftubes and introduce 50 μl dilute MP-FN with a pipette and incubate for 2hours at room temperature with continuous mixing

[0120] Centrifuge for 2 minutes at 14,000 g with NANOSEP

[0121] Discard the filtrate and resuspend MP-FN in 100 μl PBS-TWEEN 202%

[0122] Place 30 μl per sample accurately into cavity bottom

[0123] Incubate for 2 hours at 37° C. at 450 rpm

[0124] Wash 2×300 μl PBS-TWEEN 20 2%

[0125] Measuring (Time Delayed Fluorescence):

[0126] Equipment: Tecan GENios, Program Xfluor4: 10 flashes, delay 400μs, integration 400 μs.

EXAMPLE 9

[0127] Fibronectin is Coupled with Beads (Polystyrene Beads):

[0128] Some untreated samples and samples treated with proteinase K areincubated with fibronectin nanobeads

[0129] The sample is then incubated with the fibronectin beads.

[0130] Subsequently, the fibronectin beads are washed and resuspended ina reaction buffer

[0131] The resuspended beads are incubated with a monoclonalfluorescence-labelled antibody (against PrP)

[0132] After incubation, the fluorescence signals are measured in a flowcytometre.

EXAMPLE 10

[0133] Binding of PrPsc in Microtiter Plates Coated with Fibronectin

[0134] 1. The microtiter plate is coated with fibronectin

[0135] 2. The sample is incubated in the microtiter plate. Thepathological form of PrP (PrPsc) adheres to the fibronectin and thenormal form (PrPc) does not remains dissolved in the reaction mixture.

[0136] 3. Following the wash process, the europium nanobeads coupledwith monoclonal antibody (against PrP) are pipetted into the microtiterplate and incubated.

[0137] 4. Following the wash process, fluorescence is measured (timeresolution) and the concentration of PrPsc determined.

[0138] A detailed procedure is as follows, for example:

[0139] Coat a Plate (Nunc. Fluoro-Nunc modules F16 Maxi-Sorp. Cat. No.47515):

[0140] Dilute FN 2.5 μg/ml in PBS (Pierce, cat. No. 28374) pH 7.2,introduce 50 μl/well with a pipette

[0141] Incubate for 1 hour at room temperature

[0142] Remove by suction

[0143] Wash plate carefully 3 times with 300 μl distilled water.

[0144] Antigen Incubation:

[0145] Introduce sample treated with 30 μl proteinase K with a pipetteinto the cavities

[0146] Incubate for 1.5 hours at room temperature—450 rpm

[0147] MP-3F4 Incubation:

[0148] Wash once with 200 μl PBS-TWEEN 20 2%

[0149] Dilute MP 1:296 in PBS-TWEEN 20 2%

[0150] Thoroughly mix the dilution

[0151] Pipette 30 μl accurately onto cavity bottom

[0152] Incubate for 4 hours at 30° C.—shake at 450 rpm

[0153] Wash twice with 200 μl PBS Tween20 2%

[0154] Measuring:

[0155] Equipment: Tecan GENios, program XFluor4: 10 flashes, delay 400μs, integration 400 μs.

EXAMPLE 11

[0156] Binding of PrPsc to Europium Nanoparticles Via Lipoprotein A.

[0157] For procedure compare Example 9 (instead of fibronectin,lipoprotein A was used. Apolipoprotein A can also be used).

EXAMPLE 12

[0158] Binding of PrPsc to Polystyrene Beads Coated with Lipoprotein A.

[0159] For procedure compare example 10 (instead of fibronectin,lipoprotein A was used. Apolipoprotein A can also be used).

EXAMPLE 13

[0160] Binding of PrPsc in Microtiter Plates Coated with Lipoprotein A.

[0161] For procedure compare example 11 (instead of fibronectin,lipoprotein A was used. Apolipoprotein A can also be used).

1. In vitro process for detecting BSE pathogens (PrPsc) in a sample,wherein (a) the sample is treated with proteinase K, (b) the sample thustreated is brought into contact with a PrPsc-binding principle which isbound to a solid carrier (c) the binding principle bound to the solidcarrier is separated from the sample and the BSE pathogen (PrPsc) which,if necessary, is bound to the binding principle and originates from thesample is detected, characterised in that (d) the binding principle (ii)is selected from the group consisting of: a glycosaminoglycan,fibronectin or lipoprotein A.
 2. Process according to claim 1, wherein aPrPsc-specific monoclonal antibody is used for detection (c).
 3. Processaccording to claim 1 wherein the carrier from stage (b) is present inthe labelled state for the detection (c) and, following depletion fromthe sample by binding to a second binding principle, the correspondinglabelling is detected by the corresponding detection.
 4. Processaccording to one of the preceding claims, wherein the glycosaminoglycanis heparan sulphate, heparin, hyaluronic acid, chondroitin sulphate,dermatan sulphate, keraton sulphate or Congo Red.
 5. Process accordingto one of the preceding claims, wherein the binding principle is anartificially produced sulphonated glycosaminoglycan.
 6. Processaccording to one of the preceding claims 1, wherein the sampleoriginates from the human or animal body or from plants.
 7. Processaccording to claim 5 wherein the sample is a blood sample, tissue sampleor a body liquid such as urine, milk, liquor or saliva.
 8. Processaccording to claim 5 wherein the sample is thrombic material subjectedto lysis. 9 Process according to one of claims 1 to 4 wherein the sampleis a soil sample.
 10. Process according to one of the preceding claimscharacterised in that a microcarrier (bead) is used as solid carrier.11. Process according to one of the preceding claims characterised inthat the antibody used for the detection is coupled to a nanobead with adiameter of 10-100 nm which contains europium and in which the BEpathogen (PrPsc) which, if necessary, is bound to the binding principleand originates from the sample, is detected by means of time-delayedfluorescence.
 12. Process according to one of the preceding claimscharacterised in that the binding of the PrPsc to the binding principlecan be detected by time delayed fluorescence of nanobeads containingeuropium bound to the binding principle.
 13. Process according to one ofthe claims detailed above characterised in that microtiter plates areused as solid carrier.
 14. Process according to one of the claimsdetailed above characterised in that a sintered glass filter is used assolid carrier.
 15. Diagnostic system in the form of a kit for theexecution of the process for detecting BSE pathogens (PrPsc) accordingto one of the preceding claims, comprising at least the bindingprinciple bound to a solid carrier.
 16. Diagnostic system according toclaim 13 additionally comprising a proteinase, preferably proteinase K.17. Diagnostic system according to claim 13 or claim 14 additionallycomprising a specific antibody to PrPsc and, if necessary, a secondantibody directed against the specific antibody.